Field of the Invention
The invention relates to the field of biological transporter proteins and modulation of these proteins. This invention relates to a novel method to detect and measure the interaction of a test agent with mammalian phosphatidylcholine (PC) export transport and/or formation activity.
Description of Related Art
P-glycoproteins (P-gp) are 170-kDa glycosylated membrane proteins that actively transport and export a wide range of substrates out of cells (Smith, A., et al. [2000]. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping. J Biol Chem, 275[31], 23530-23539. p 23531.). Two P-gp genes have been identified in humans that encode for the multidrug-resistant 1 and 3 P-glycoproteins (MDR1 and MDR3). Multidrug-resistant 3 P-glycoprotein is also referred to as multidrug resistance protein 3 and is used interchangeably. MDR1 and MDR3 are members of the ATP-binding cassette (ABC) transporter family and are also named ATP-binding cassette proteins B1 (ABCB1) and B4 (ABCB4), respectively (Smith, id., p 23531.; Yoshikado, T., et al. [2011]. Itraconazole-induced cholestasis: involvement of the inhibition of bile canalicular phospholipid translocator MDR3/ABCB4. Mol Pharmacol, 79(2), 241-250. p 241; Zhao, Y., et al. [2015]. ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner. J Lipid Res. doi:10.1194/jlr.M056622. p 3.). MDR1 is associated with the export of several drugs and is involved in the resistance that cancer cells display to several anticancer agents (Smith, id., p 23531.; Yoshikado, id., p 241.). Based on the process by which MDR3 translocates PC from the inner to outer leaflet of cellular membranes, MDR3 is also described as a type of floppase protein and is involved in maintaining cannicular membrane integrity (Groen, A., et al. [2011]. Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity. Gastroenterology, 141(5), 1927-1937. p 1927.). MDR3 appears to play a lesser role in drug transport compared with MDR1, although more examples continue to be found (Smith, id., p 23531.).
MDR3 (ABCB4) is a 1279 amino acid protein that is divided into two homologous halves, each of which contains six transmembrane helices (TMHs) and a cytoplasmic nucleotide-binding fold (NBF) (Morita, S. Y., & Terada, T. [2014]. Molecular mechanisms for biliary phospholipid and drug efflux mediated by ABCB4 and bile salts. Biomed Res Int, 2014: 954781. doi: 10.1155/2014/954781. p 1.). MDR1 is found in various tissues, including the liver, kidney, intestinal mucosa, and capillary endothelial cells at the blood-brain barrier, while MDR3 is expressed mainly in hepatic tissue, although low levels of MDR3 mRNA can be found in the adrenal gland, muscle, tonsil, spleen, placenta, testis, and ileum (Morita & Terada, id., p 1.).
Bile formation is one of the most essential functions of the liver, and several ATP-binding cassette (ABC) transporters are known to be involved in the biliary secretion of biliary lipids, organic solutes, and xenobiotics (Yoshikado, id., p 241.). Biliary secretion of bile salts and phospholipids, essential components of biliary micelles, are mediated by the bile salt export pump (BSEP also known as ABCB11) and by MDR3, respectively, and their genetic dysfunction can lead to severe cholestatic diseases (Yoshikado, id., p 241.). Dysfunction of MDR3 results in a lack of phospholipids and a surplus of bile salts in primary bile, and this compositional imbalance causes damage to biliary canaliculi, leading to chronic and progressive liver diseases such as intrahepatic cholestasis and low phospholipid-associated cholelithiasis (LPAC) syndrome (Zhao, id., p 3.).
In Abcb4 knockout mice models, biliary secretion of phospholipids is negligible and excretion of cholesterol is reduced (Morita & Terada, id., p 3.). In Abcb4 heterozygous mice the secretion rate of phospholipids is reduced by about 30 to 50 percent, while cholesterol secretion remains similar to that found in wild-type mice (Morita & Terada, id., p 3.). Mixed micelles of bile salts and phospholipids have a much higher capacity to take up cholesterol than simple bile salt micelles (Morita & Terada, id., p 3.). In a vesicle model prepared from a yeast mutant expressing the mouse Abcb4 gene, the addition of the bile salt taurocholate increases the ABCB4 transporter activity for phospholipid (Morita & Terada, id., p 6.). The phospholipid efflux mediated by ABCB4 is increased with increasing concentrations of taurocholate and shows concentration dependence from 0.2 mM to 1 mM taurocholate (Morita & Terada, id., p 6.).
Mutations in the human MDR3 (ABCB4) gene are associated with a wide spectrum of hepatic injury phenotypes, ranging from progressive familial intrahepatic cholestasis type 3 (PFIC3) to adult cholestatic liver disorders (Morita & Terada, id., p 3.). PFIC3 is characterized by high γ-glutamyl transpeptidase and early onset of persistent cholestasis that progresses to cirrhosis and liver failure before adulthood (Morita & Terada, id., p 3.). In many cases of PFIC3, liver transplantation is the only therapy. The biliary phospholipid level in a PFIC3 patient is dramatically decreased despite the presence of bile salts (Morita & Terada, id., p 3.).
Defects in the MDR3 transporter are associated with intrahepatic cholestasis of pregnancy (ICP), LPAC, and primary biliary cirrhosis (Morita & Terada, id., p 3.). ICP is a reversible form of cholestasis in the third trimester of pregnancy and is rapidly ameliorated after childbirth. LPAC is characterized by intrahepatic hyperechoic foci, intrahepatic sludge, or microlithiasis (Morita & Terada, id., p 3.). The absence of biliary phospholipids may lead to the destabilization of micelles and promote the lithogenicity of bile with the crystallization of cholesterol (Morita & Terada, id., p 3.). The association between cholangiocarcinoma, a rare malignant tumor of the biliary tract, and ABCB4 mutations has recently been reported (Morita & Terada, id., p 3.). Chronic biliary inflammation may increase cholangiocyte turnover, leading to the growth of altered cholangiocytes and increased susceptibility to cholangiocarcinoma (Morita & Terada, id., p 3.).
Hepatocytes: Hepatic parenchymal cells, or hepatocytes, are polyhedral or spherical in nature and account for approximately 60% of the cells in the liver; they represent 80% or more of the total liver volume (de la Iglesia, F. [1999]. Morphofunctional aspects of hepatic structure. In: Handbook of Drug Metabolism, Woolf, T. F., editor, New York: Dekker. p 83.). Hepatocytes are polar in nature, and one skilled in the art would recognize what is termed an apical (canalicular) membrane or domain and a basolateral (blood or sinusoidal domain) membrane or domain. The hepatocyte basolateral membrane or domain is involved in the uptake of drugs and xenobiotics into the cell, while the apical membrane or domain provides a route for intracellular produced bile salts to be excreted or transported into bile flow and eventually to the common bile duct for secretion into the intestine.
Hepatocytes have specialized transport systems or transcellular transporters located at the basolateral membrane and the apical membrane (Morgan, R. E., et al. [2010]. Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol Sci, 118(2), 485-500. p 485.). These hepatobiliary transporters maintain liver homeostasis by regulating intracellular exposure to endobiotic and xenobiotic chemicals. Transport systems comprising specific transporter proteins have been extensively investigated. Transporters at the basolateral membrane are involved in hepatocellular uptake of various substrates from the blood and sinusoids, elimination to the blood and sinusoids, or both, depending on the transporter. Transporters on the apical membrane, however, are exclusively efflux transporters, mediating secretion of various substrates into the bile flow including bile acids, phospholipids, and salts (Morgan, id., p 485.).
Drug-Induced Liver Injury (DILI): Drug-induced liver injury encompasses a spectrum of clinical disease ranging from mild biochemical abnormalities to acute liver failure (Hussaini, S. H. & Farrington, E. A. [2007]. Idiosyncratic drug-induced liver injury: an overview. Expert Opin Drug Saf, 6(6), 673-684. Abstract.). Most frequently, the underlying mechanism of DILI is poorly understood. In some cases of DILI, the liver injury is categorized as idiosyncratic—unknown etiology (Lee, W. M. [2003]. Drug-induced hepatotoxicity. N Engl J Med, 349(5), 474-485. Abstract; Wolf, K. K., et al. [2010]. Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport. Toxicol In Vitro, 24(1), 297-309. p 2.). The incidence of DILI-induced hepatotoxicity in clinically marketed drugs is relatively rare, ranging from 1 in 5,000 to 1 in 10,000 or less; particularly infrequently does DILI result in severe liver injury leading to irreversible liver failure that can be fatal or require liver transplantation. Despite this low incidence, DILI is a major cause of removal of approved drugs from the United States market resulting in removal of clinically significant therapeutics from patients in need of such therapy (Wolf, id., p 1.; U.S. Department of Health and Human Services, Food and Drug Administration (FDA), Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for Industry: Drug-induced liver injury—premarketing clinical evaluation. Silver Springs, Md.: FDA, July 2009. p 1, Introduction.). Further consequences of DILI include class action lawsuits against the innovator company (with multimillion dollar settlements), and the addition of time, expense, and uncertainty to the drug discovery and development process.
The incidence of DILI in approved drugs is rare because modern drug development requires extensive preclinical testing of drug candidates and subsequent clinical trials. Drug candidates that display toxic potential are usually removed from development and never reach the market (FDA, id.). Nevertheless, some drugs that reach the market do produce DILI. Reasons for this may involve the relatively infrequent occurrence or nature of an adverse event and the fact that clinical trials are conducted in a closely controlled patient environment with a limited number of subjects for a limited time. Following marketing approval, the number of individuals who are administered a therapeutic agent will be much greater, periods of treatment may be much longer, and patients are less well monitored. Individuals display a wide variability in hepatic function and can differ greatly with respect to inherent hepatic metabolic function, environmental factors, and use of other medications. Risk factors for DILI include age, sex, and genetic polymorphisms of drug-metabolizing enzymes such as cytochrome P450. In patients with human immunodeficiency virus, the presence of chronic viral hepatitis increases the risk of antiretroviral therapy hepatotoxicity (Wolf, id.; Hussaini, id. Abstract.).
The relatively low incidence rate of DILI creates difficulties in detecting and diagnosing it, both in lack of tests and in numbers of patients needed to be tested. There is no clinical finding that indicates the presence of DILI with certainty, including liver biopsy. Because DILI may simulate a variety of known liver diseases, the histopathologic picture frequently is reported to be “compatible with” the clinical and laboratory information available, but is often not diagnostic. In fact, the diagnosis of DILI is one of exclusion, in which sufficient clinical information must be gathered to rule out other possible causes of abnormal findings. This kind of diagnosis requires collecting considerable data at the time of the acute clinical situation, a process that is frequently inefficiently and haphazardly done, and therefore available information is often inadequate to establish the likelihood of drug causality with a reasonable degree of confidence (FDA, id., pps 3-7.).
In most controlled clinical trials, monitoring is done to detect hepatic injury by serum enzyme (typically aminotransferase) activity increase. Because risks associated with a new drug are unknown, caution dictates that stopping rules be used to limit liver damage during a trial. For safety reasons, the drug may be stopped before the full implication of its possible toxicity can be determined. Extrapolation of such potentially incomplete data is therefore often used to predict the likelihood of future severe toxicity of the drug in clinical use.
In order to interpret data from patients exposed to drugs in clinical trials, a hierarchy of findings that indicate progressively severe liver injury is used, beginning with serum aminotransferase activity as the most frequently abnormal and most sensitive test (FDA, id.). In clinical trials of new drugs, 15% or more of study patients may demonstrate mild elevations of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) activity. The threshold required to consider either more frequent monitoring of blood levels or stopping the drug is variously placed at twice the upper limit of the normal (ULN) or reference range (2×ULN), at 3×ULN, or at 5×ULN. Monitoring is typically performed on a monthly basis but may be increased to biweekly or weekly checking if elevations in serum enzyme levels are noted. According to the FDA Guidance on Drug-Induced Liver Injury:
Discontinuation of Treatment should be Considered when:
ALT or AST>8×ULN
ALT or AST>5×ULN for more than 2 weeks
ALT or AST>3×ULN and (TBL>2×ULN or INR>1.5)
ALT or AST>3×ULN with the appearance of fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and/or eosinophil (>5%)
ALT—Alanine aminotransferase; AST—Aspartate aminotransferase; TBL—Total bilirubin levels; INR—increased plasma thrombin time.
U.S. Department of Health and Human Services, Food and Drug Administration (FDA), Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for Industry: Drug-induced liver injury—premarketing clinical evaluation. Silver Springs, Md.: FDA, July 2009. Page 10.
Levels of 10×ULN typically mandate immediate cessation of a drug or agent and are considered serious signals, but still do not represent true tests of liver function. Yet great difficulties persist in accurately determining, when abnormalities are seen, whether they are caused by DILI or by some other disorder (FDA, id., pps 3-7, 10.).
Even modest increases of serum total bilirubin concentration may represent the beginning of reduced bilirubin excretion capacity, provided that Gilbert's syndrome and other unrelated causes of bilirubin elevation can be excluded. It is a function of the liver to clear plasma of bilirubin and excrete it into the bile. The late Hyman Zimmerman, in 1978 and again in 1999, after a careful review of clinical trials and literature reports, proposed that the appearance of jaundice in association with drug-induced hepatocellular injury indicated possible mortality in about 10 to 50 percent of patients with that combination (FDA, id., p 4.).
Another commonly done test, the blood prothrombin time (or its derivative, Internationalized Ratio, INR) may be useful as a liver function test of protein synthesis. In acute liver failure caused by acetaminophen overdose, increases in INR may precede rises in total bilirubin levels. Thus, only a small decrement in liver function in pre-approval trials may provide a signal that additional and more severe cases may occur when larger numbers of patients are exposed. The full impact of this may not be realized until after a drug's approval for clinical use and marketing.
The condition of cholestasis occurs when bile and bile fluids cannot flow from the hepatocytes to the duodenum. The accumulation of bile salts in hepatocytes can lead to cellular apoptosis, necrosis, and mitochondrial dysfunction (Wolf et al., id., p 298.). Cholestasis may result from physical obstructions such as gallstones or tumors, or from metabolic disorders as a result of drugs interfering with BSEP, MDR3, and other transporters.
MDR3 and DILI: MDR3 and BSEP are potentially important targets for drug-induced liver injury. MDR3 is able to transport a number of MDR1 substrates (e.g., digoxin, paclitaxel, and vinblastine), although the contribution of MDR3 clinically is probably less important than other transporters such as MDR1. Verapamil, cyclosporine, and vinblastine are able to inhibit MDR3, explaining why these drugs could adversely affect canalicular phosphatidylcholine secretion (Lang, T., et al. [2006] Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11). Drug Metabolism and Disposition, 34(9), 1582-1599. p 1582.).
In contrast to xenobiotic transporters such as MDR1, which control the access of substrates to pharmacological sanctuaries, changes in MDR3 and BSEP transport function may have their greatest impact on occurrence, pattern, and prognosis of cholestatic liver injury. Furthermore, they may modulate the individual sensitivity to drug-mediated inhibition of MDR3 and BSEP transport and, consequently, the individual sensitivity to DILI (Lang, id., p 1598.).
MDR3 and drug interactions: ABCB4-expressing BRO human melanoma cells exhibit no resistance to a range of drugs including vincristine, colchicine, etoposide, daunorubicin, doxorubicin, actinomycin D, and gramicidin D (Morita & Terada, id., p 6.). Vesicles prepared from expression of ABCB4 in yeast confer resistance to aureobasidin A, an antifungal cyclic depsipeptide antibiotic, which is overcome by vinblastine, verapamil, and cyclosporine A (Morita & Terada, id., p 6.). Smith et al. have reported that polarized monolayers of ABCB4-expressing LLC-PK1 cells show an increased directional transport of several ABCB1 substrates, such as digoxin, paclitaxel, daunorubicin, vinblastine, and ivermectin, and that the transport rate of these drugs, except for paclitaxel, is lower in ABCB4-expressing cells than in ABCB1-expressing cells (Smith, id., p 23536.). Furthermore, ABCB4-dependent transport of digoxin is inhibited by ABCB1 reversal agents, cyclosporine A, valspodar, and verapamil (Morita & Terada, id., p 6.). In addition, expression of ABCB1 or ABCB4 in HEK293 cells decreases the accumulation of rhodamine 123 and rhodamine 6G, and these reductions are more marked in ABCB1-expressing cells than in ABCB4-expressing cells (Morita & Terada, id., p 6.). The accumulation of BODIPY-verapamil in HEK293 cells is strikingly reduced by ABCB1 expression but is not altered by ABCB4 expression, indicating that BODIPY-verapamil is not a transport substrate of ABCB4 but an inhibitor of the ABCB4-mediated phospholipid efflux (Morita & Terada, id., p 6.). These findings suggest that ABCB4 cannot cause multidrug resistance due to the low rates of ABCB4-mediated export of drugs compared with ABCB1-mediated export. The nonphospholipid substrates may have lower affinities for ABCB4 than ABCB1 and/or compete with membrane PC for binding to ABCB4. Furthermore, the addition of taurocholate has no effect on the ABCB4-mediated efflux of rhodamine 123 and rhodamine 6G, which may be attributed to sufficient solubility of these substrates in the aqueous medium (Morita & Terada, id., p 6.).
The ABCB4-mediated secretion of PC is enhanced by the activation of protein kinase A or C and is decreased by the inhibition of these kinases (Morita & Terada, id., p 6.). Itraconazole, an antifungal agent, is known to cause drug-induced cholestasis (DIC) and is associated in a rat model of cholestasis with a significant decrease in biliary phospholipids (Morita & Terada, id., p 6.). In additional studies involving ABCB4-mediated efflux of carbon-14 radiolabeled PC from LLC-PK1 cells, the presence of itraconazole decreased the efflux of PC (Morita & Terada, id., p 6.).
Expression of MDR3 in Sf9 Cells: The MDR3 gene is cloned into baculovirus and expressed in Sf9 insect cells by methods that one skilled in the relevant art would readily understand (Smith, id., p 235635.). Vesicle membranes are prepared from the Sf9 insect cells expressing MDR3 following cell disruption homogenizations and centrifugations (Smith, id., p 23535.). Vesicle preparations are used in experiments to measure PC transport utilizing fluorescence or radiolabeled substrates.
LLC-PK1 cells derived from a pig kidney epithelial cell line are transfected with the MDR3 gene and cultured for use in drug transport assays (Smith, id., p 23534.). Transfected LLC-PK1 cells are used in directional transport experiments. However, assays have to be repeated with various batches of cell lines to obtain useful data to compare activities (Smith, id., p 23533.).
The current MDR3 export transport assays have many issues including: (1) complexity and variability in preparing inside-out vesicles from transfected insect cell lines; (2) physiological relevance of vesicles prepared from insect cell lines; (3) transfected mammalian cells of different complexities and variable expression and/or activities; (4) requirement of radiolabeled phosphatidylcholine or fluorescence phospholipid precursors; (5) inability to assess the indirect effect of a test agent's metabolism on MDR3 transport; and (6) assays do not allow exploration of interactions under physiological conditions in which bile salts and other transporters are present. In general, less is known about MDR3 because of the difficulty of establishing a robust activity assay (Ellinger, P., et al. [2013]. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris. PLoS One, 8(4), e60620. p 2.).